Can Suillus granulatus (L.) Roussel be classified as a functional … · 2018. 6. 13. · 4 Suillus...
Transcript of Can Suillus granulatus (L.) Roussel be classified as a functional … · 2018. 6. 13. · 4 Suillus...
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Can Suillus granulatus (L.) Roussel be classified as a functional food?
Filipa S. Reisa,b,c, Dejan Stojkovićd, Lillian Barrosa, Jasmina Glamočlijad, Ana Ćirićd,
Marina Sokovićd, Anabela Martinsa, M. Helena Vasconcelosc,e, Patricia Moralesb, Isabel
C.F.R. Ferreiraa*
aMountain Research Center (CIMO), ESA, Polytechnic Institute of Bragança, Campus
de Santa Apolónia, Ap. 1172, 5301-855 Bragança, Portugal.
bDpto. Nutrición y Bromatología II, Facultad de Farmacia, Universidad Complutense
de Madrid (UCM), Pza Ramón y Cajal, s/n, E-28040 Madrid, Spain.
cCancer Drug Resistance Group, IPATIMUP – Institute of Molecular Pathology and
Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto,
Portugal.
dUniversity of Belgrade, Department of Plant Physiology, Institute for Biological
Research “Siniša Stanković”, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia.
eLaboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy
of the University of Porto, Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto,
Portugal.
* Corresponding author. Tel.: +351 273 303219; fax: +351 273 325405.
E-mail address: [email protected] (I.C.F.R. Ferreira).
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ABSTRACT
The present work outlines a detailed chemical characterization of Suillus granulatus
species, besides the antioxidant and antimicrobial properties of their methanolic
extracts. The study was carried out with samples drawn from Portugal and Serbia in
order to prove that though mushrooms are strongly influenced by the environment in
which they develop, they have a specific chemical profile that can be typical of their
genus/species. The studied species proved to be healthy foods, low in fat and rich in
protein and carbohydrates, with mannitol and trehalose being the main free sugars
detected. They also proved to be a source of organic and phenolic acids, as well as
mono- and polyunsaturated fatty acids and tocopherols. The Serbian samples revealed
higher antioxidant and antimicrobial potential. Accordingly, we find that the S.
granulatus species is likely to be considered a functional food, since it is a source of
nutraceutical and biologically active compounds.
Keywords: Chemical characterization; nutraceuticals, bioactive compounds; antioxidant
potential; antimicrobial activity.
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Introduction
Due to current daily habits, busy lifestyles and the consequent increase in several
chronic diseases, there is a need to develop alternative food sources which while
satisfying consumer demand, also have beneficial effects on health. Functional foods
appear in this context. Because of the complexity of the term “functionality”, no agreed
and universally accepted definition for this group of food currently exists.1 Furthermore,
functional foods have been considered as a concept rather than as a well-defined group
of food products. The European Commission’s Concerted Action on Functional Food
Science in Europe (FUFOSE) stated that functional food is “a food that beneficially
affects one or more target functions in the body beyond adequate nutritional effects in a
way that is relevant to either an improved state of health and well-being and/or
reduction of risk of disease. It is consumed as part of a normal food pattern. It is not a
pill, a capsule or any form of dietary supplement”.2 A functional food can be a natural
food or a food to which a component has been added or removed by technological or
biotechnological means.3 People used to associate the term “functional food” to
technological or genetically-modified food. Indeed, the enrichment or addition of
functional ingredients, as well as the removal of some compounds with negative effects
induced by food technology procedures, or the alteration of food products to enhance
their nutritional value using genetic modifications, constitute a category of functional
foods.4 However, since the definition of functional foods is related with the beneficial
effect that goes beyond those of traditional nutrients, if it is scientifically proven that
certain compounds present in some food reduces for example, the risk of developing a
certain illness, this food can be considered a functional food and this may include
natural products.4
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Suillus granulatus (L.) Roussel, known as the “weeping bolete”, is an edible mushroom
with a white, soon yellowish and non-staining flesh. It has a mild to slightly fragrant
odour and tastes mild.5 Although this species (as all the Suillus species) is not one of the
most consumed as a delicacy, such as truffles or morels, it is widely harvested and
consumed by the general population, particularly those who traditionally practice
mushroom picking. Because of its mild taste, it is often mixed with other species to
improve taste / flavour attributes.6
Some reports involving this species can be found. Some are ecological studies which
tried to prove that this ectomycorrhizal fungus could utilise litter as a source of nutrients
and therefore reduce the negative effects of litter accumulation in forest ecosystems.7
Another study describs a β-carboline compound isolated from S. granulatus with a weak
anti-HIV-1 activity.8 Concerning the chemical characterization of this species, there are
few reports published regarding the fatty acid,9 organic acid phenolic compound
compositions and antioxidant activity.10
The present work intends to take the first step towards classifying Suillus granulatus as
a functional food, providing a detailed chemical analysis of the species which proves
that this is a source of nutraceuticals and/or biologically active molecules. By
comparing mushrooms collected from different locations it was intended to analyse the
different chemical profiles, in order to confirm whether they remain unaltered
depending on the surrounding environment.
Experimental
Mushroom species
Suillus granulatus (L.) Roussel wild samples were collected in Bragança (Northeast of
Portugal) and in Lipovica Forest, near Belgrade (Serbia), in the autumn of 2012. The
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authentications were undertaken at the Polytechnic Institute of Bragança and Institute
for Biological Research, Belgrade. Voucher specimens were deposited at the herbarium
of the School of Agriculture of the Polytechnic Institute of Bragança, Portugal, and at
the Fungal Collection Unit of the Mycological Laboratory, Department for Plant
Physiology, Institute for Biological Research “Siniša Stanković”, Belgrade, Serbia,
respectively.
All samples were lyophilised (FreeZone 4.5 model 7750031, Labconco, Kansas City,
MO, USA), reduced to a fine dried powder (20 mesh), mixed to obtain homogenous
samples and stored in a desiccator, protected from light, until further analysis.
Standards and Reagents
Acetonitrile 99.9%, n-hexane 95% and ethyl acetate 99.8% were of HPLC grade from
Fisher Scientific (Lisbon, Portugal). The fatty acids methyl ester (FAME) reference
standard mixture 37 (standard 47885-U) was purchased from Sigma (St. Louis, MO,
USA), as well as other individual fatty acid isomers, sugar (D(-)-fructose, D(-)-
mannitol, D(+)-raffinose pentahydrate, and D(+)-trehalose), tocopherol (α-, β-, γ-, and
δ-isoforms) and organic acid (oxalic, quinic, malic, citric and fumaric acid) standards.
Racemic tocol, 50 mg/mL, was purchased from Matreya (Pleasant Gap, PA, USA). 2,2-
Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA,
USA). Phenolic standards (gallic, p-hydroxybenzoic and cinnamic acids) and trolox (6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma
(St. Louis, MO, USA). Mueller–Hinton agar (MH) and malt agar (MA) were obtained
from the Institute of Immunology and Virology, Torlak (Belgrade, Serbia).
Dimethylsulfoxide (DMSO), (Merck KGaA, Darmstadt, Germany) was used as a
solvent. Methanol and all other chemicals and solvents were of analytical grade and
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purchased from common sources. Water was treated in a Milli-Q water purification
system (TGI Pure Water Systems, Greenville, SC, USA).
Chemical characterization
Macronutrients composition
The samples were analysed for their nutritional chemical composition (protein, fat,
carbohydrate and ash) through standard procedures.11 The crude protein content (N ×
4.38) of the samples was estimated by the macro-Kjeldahl method; crude fat was
determined by extracting a known weight of powdered sample with petroleum ether,
using a Soxhlet apparatus; ash content was determined by incineration at 600±15 ºC.
Total carbohydrate was calculated by difference. Energy was calculated according to the
following equation: Energy (kcal) = 4 × (g protein + g carbohydrate) + 9 × (g fat).
Hydrophilic compounds
Free sugars. Free sugars were determined by a high performance liquid chromatograph
(HPLC) system consisting of an integrated system with a pump (Knauer, Smartline
system 1000, Berlin, Germany), degasser system (Smart line manager 5000) and an
auto-sampler (AS-2057 Jasco, Easton, MD, USA), coupled to a refraction index
detector (RI detector Knauer Smartline 2300) as previously described by the
authors.12,13 Sugars identification was undertaken by comparing the relative retention
times of sample peaks with standards. Data were analyzed using Clarity 2.4 Software
(DataApex, Podohradska, Czech Republic). Quantification was based on the RI signal
response of each standard, using the internal standard (IS, raffinose) method and by
using calibration curves obtained from the commercial standards of each compound.
The results were expressed in g per 100 g of dry weight.
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Organic acids. Organic acids were determined following a procedure previously
described by the authors.12,13 Analysis was performed using a Shimadzu 20A series
UFLC (Shimadzu Corporation, Kyoto, Japan). Separation was achieved on an
SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C18 column (5 µm, 250
mm × 4.6 mm i.d) thermostatted at 35 ºC. The elution was performed with sulphuric
acid 3.6 mM using a flow rate of 0.8 mL/min. Detection was carried out in a DAD,
using 215 nm and 245 nm (for ascorbic acid) as preferred wavelengths. The organic
acids found were quantified by comparison of the area of their peaks recorded at 215
nm with calibration curves obtained from commercial standards of each compound. The
results were expressed in g per 100 g of dry weight.
Phenolic acids and related compounds. Phenolic acid determination was performed
using a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu
Corporation, equipment described above) as previously described by the authors.12,13
Detection was carried out in a photodiode array detector (PDA), using 280 nm as the
preferred wavelength. The phenolic acids were quantified by comparison of the area of
their peaks recorded at 280 nm with calibration curves obtained from commercial
standards of each compound. The absence of other phenolic compounds in the samples
was confirmed using mass spectrometry. The results were expressed in mg per 100 g of
dry weight.
Lipophilic compounds
Fatty acids. Fatty acids were determined after a trans-esterification procedure as
described previously by the authors.12,13 The fatty acid profile was analyzed with a
DANI 1000 gas chromatographer (GC) equipped with a split/splitless injector and a
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flame ionization detector (FID). Fatty acid identification was made by comparing the
relative retention times of FAME peaks from samples with standards. The results were
recorded and processed using Clarity 4.0.1.7 Software (DataApex, Podohradska, Czech
Republic) and expressed in relative percentage of each fatty acid.
Tocopherols. Tocopherols were determined following a procedure previously described
by the authors.12,13 Analysis was performed by HPLC (equipment described above), and
a fluorescence detector (FP-2020; Jasco) programmed for excitation at 290 nm and
emission at 330 nm. The compounds were identified by chromatographic comparisons
with authentic standards. Quantification was based on the fluorescence signal response
of each standard, using the IS (tocol) method and by using calibration curves obtained
from commercial standards of each compound. The results were expressed in µg per100
g of dry weight.
Bioactivity evaluation
Extract preparation
The lyophilized samples (1 g) were extracted by stirring with 40 mL of methanol for 1 h
and subsequently filtered through Whatman No. 4 paper. The residue was then extracted
with 20 mL of methanol for 1 h. The combined methanolic extracts were evaporated at
40ºC (rotary evaporator Büchi R-210, Büchi, Flawil, Switzerland) to dryness and re-
dissolved in a) methanol for the antioxidant activity assays (20 mg/mL) and b) a 5%
solution of DMSO in distilled water for the antimicrobial activity assays (100 mg/mL).
Antioxidant properties
Successive dilutions were made from the stock solution and submitted to the in vitro
assays already described by Reis et al.14, to evaluate the antioxidant activity of the
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samples. The sample concentrations (mg/mL) providing 50% of antioxidant activity or
0.5 of absorbance (EC50) were calculated from the graphs of antioxidant activity
percentages (DPPH, β-carotene/linoleate and TBARS assays) or absorbance at 690 nm
(ferricyanide/Prussian blue assay) against sample concentrations. Trolox was used as a
positive control.
Folin-Ciocalteu assay. One of the extract solutions (5 mg/mL for the Portuguese sample
and 1.25 mg/mL for the Serbian sample; 1 mL) was mixed with Folin-Ciocalteu reagent
(5 mL, previously diluted with water 1:10, v/v) and sodium carbonate (75 g/L, 4 mL).
The tubes were vortex-mixed for 15 sec and allowed to stand for 30 min at 40ºC for
colour development. Absorbance was then measured at 765 nm (Analytikjena
spectrophotometer; Jena, Germany). Gallic acid was used to obtain the standard curve
and the reduction of the Folin-Ciocalteu reagent by the samples was expressed as mg of
gallic acid equivalents (GAE) per g of extract.
Ferricyanide/Prussian blue assay. The extract solutions with different concentrations
(0.5 mL) were mixed with sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and
potassium ferricyanide (1% w/v, 0.5 mL). The mixture was incubated at 50°C for 20
min and trichloroacetic acid (10% w/v, 0.5 mL) was added. The mixture (0.8 mL) was
poured in the 48 wells plate, the same with deionised water (0.8 mL) and ferric chloride
(0.1% w/v, 0.16 mL), and the absorbance was measured at 690 nm in ELX800
Microplate Reader (Bio-Tek Instruments, Inc; Winooski, VT, USA).
DPPH radical-scavenging activity. This methodology was performed using the
Microplate Reader mentioned above. The reaction mixture in each of the 96-well of the
plate consisted of one of the different concentrations of the extracts (30 µl) and
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methanolic solution (270 µL) containing DPPH radicals (6×10-5 mol/L). The mixture
was left to stand for 30 min in the dark and the absorption was measured at 515 nm. The
radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration
using the equation: %RSA=[(ADPPH−AS)/ADPPH]×100, where AS is the absorbance of the
solution containing the sample, and ADPPH is the absorbance of the DPPH solution.
Inhibition of β-carotene bleaching or β-carotene/linoleate assay. A solution of β-
carotene was prepared by dissolving β-carotene (2 mg) in chloroform (10 mL). Two
millilitres of this solution were pipetted into a round-bottom flask. The chloroform was
removed at 40°C under vacuum and linoleic acid (40 mg), Tween 80 emulsifier (400
mg), and distilled water (100 mL) were added to the flask with vigorous shaking.
Aliquots (4.8 mL) of this emulsion were transferred into test tubes containing extract
solutions with different concentrations (0.2 mL). The tubes were shaken and incubated
at 50°C in a water bath. As soon as the emulsion was added to each tube, the zero time
absorbance was measured at 470 nm. β-Carotene bleaching inhibition was calculated
using the following equation: (Absorbance after 2 h of assay/ initial absorbance) × 100.
Thiobarbituric acid reactive substances (TBARS) assay. Porcine (Sus scrofa) brains
were obtained from official slaughtered animals, dissected, and homogenized with
Polytron in an ice cold Tris-HCl buffer (20 mM, pH 7.4) to produce a 1:2 w/v brain
tissue homogenate which was centrifuged at 3000 g for 10 min. An aliquot (100 µL) of
the supernatant was incubated with the different concentrations of the sample solutions
(200 µL) in the presence of FeSO4 (10 mM; 100 µL) and ascorbic acid (0.1mM; 100
µL) at 37°C for 1 h. The reaction was stopped by the addition of trichloroacetic acid
(28% w/v, 500 µL), followed by thiobarbituric acid (TBA, 2%, w/v, 380 µL) and the
mixture was then heated at 80°C for 20 min. After centrifugation at 3000 g for 10 min
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to remove the precipitated protein, the colour intensity of the malondialdehyde (MDA)-
TBA complex in the supernatant was measured by its absorbance at 532 nm. The
inhibition ratio (%) was calculated using the following formula: Inhibition ratio (%) =
[(A−B)/A] × 100%, where A and B were the absorbance of the control and the sample
solution, respectively.
Antimicrobial properties
Successive dilutions were made from the DMSO:water stock solution and submitted to
antibacterial and antifungal assays.
Antibacterial activity. The following Gram-negative bacteria: Escherichia coli (ATCC
35210), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC
13311), Enterobacter cloacae (ATCC 35030), and Gram-positive bacteria:
Staphylococcus aureus (ATCC 6538), Bacillus cereus (clinical isolate), Micrococcus
flavus (ATCC 10240), and Listeria monocytogenes (NCTC 7973) were used. The
microorganisms were obtained from the Mycological Laboratory, Department of Plant
Physiology, Institute for Biological Research “Siniša Stanković”, University of
Belgrade, Serbia.
The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were
determined by the microdilution method.15 Briefly, a fresh overnight culture of bacteria
was adjusted by the spectrophotometer to a concentration of 1×105 CFU/mL. The
requested CFU/mL corresponded to a bacterial suspension determined in a
spectrophotometer at 625 nm (OD625). Dilutions of inoculate were cultured on solid
medium to verify the absence of contamination and check the validity of the inoculum.
Different solvent dilutions of methanolic extract were carried out over the wells
containing 100 µL of Tryptic Soy Broth (TSB) and thereafter, 10 µL of inoculum was
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added to all of the wells. The microplates were incubated for 24h at 37°C. The MIC of
the samples was detected following the addition of 40 µL of iodonitrotetrazolium
chloride (INT) (0.2 mg/mL) and incubation at 37°C for 30 min. The lowest
concentration which produced a significant inhibition (around 50%) of the growth of the
bacteria in comparison with the positive control was identified as the MIC. The
minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of
various bacteria to tested extract were also determined by a colorimetric microbial
viability assay based on reduction of a INT colour and compared with positive control
for each of the bacterial strains.16,17 MBC was determined by serial sub-cultivation of 10
µL into microplates containing 100 µL of TSB. The lowest concentration which showed
no growth after this sub-culturing was read as the MBC. Standard drugs, namely
streptomycin and ampicillin were used as positive controls. 5% DMSO was used as
negative control.
Antifungal activity. For the antifungal bioassays, the following microfungi were used:
Aspergillus fumigatus (1022), Aspergillus ochraceus (ATCC 12066), Aspergillus
versicolor (ATCC 11730), Aspergillus niger (ATCC 6275), Penicillium funiculosum
(ATCC 36839), Penicillium ochrochloron (ATCC 9112), Trichoderma viride (IAM
5061), and Penicillium verrucosum var. cyclopium (food isolate). The organisms were
obtained from the Mycological Laboratory, Department of Plant Physiology, Institute
for Biological Research “Siniša Stanković´”, Belgrade, Serbia. The micromycetes were
maintained on malt agar (MA) and the cultures were stored at 4°C and subcultured once
a month.18
The fungal spores were washed from the surface of agar plates with sterile 0.85% saline
containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline
to a concentration of approximately 1.0×105 in a final volume of 100 µL/well. The
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inoculums were stored at 4°C for further use. Dilutions of the inoculums were cultured
on solid MA to verify the absence of contamination and to check the validity of the
inoculum.
Minimum inhibitory concentrations (MICs) determination was performed by a serial
dilution technique using 96-well microtitre plates. The investigated extract was
dissolved in 5% solution of DMSO and added to broth malt medium with fungal
inoculum. The microplates were incubated for 72 h at 28°C. The lowest concentrations
without visible growth (at the binocular microscope) were defined as MIC. The
minimum fungicidal concentrations (MFCs) were determined by serial sub-cultivation
of 2 µL in microtitre plates containing 100 µL of malt broth per well and further
incubation for 72 h at 28°C. The lowest concentration with no visible growth was
defined as the MFC, indicating 99.5% killing of the original inoculum. 5 % DMSO was
used as a negative control, while bionazole and ketokonazole were used as positive
controls.
Statistical analysis
Three samples were used and all assays were carried out in triplicate. The results are
expressed as mean values and standard deviation (SD). The results were analysed using
one-way analysis of variance (ANOVA) followed by Tukey’s HSD Test with α = 0.05.
This analysis was carried out using SPSS v. 20.0 program.
Results and discussion
Chemical composition
As referred above, all assays were performed for both samples (Portuguese and Serbian)
and the results were compared with each other. Concerning the nutritional value of the
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samples (Table 1), the Portuguese one revealed higher protein (14.78 g/100 g dw) and
fat contents (3.74 g/100 g dw); however, the highest content of carbohydrates was
detected in the Serbian sample (81.42 g/100 g dw). The Portuguese species also
revealed the highest energy contribution (386.74 kcal/100 g dw) compared with the
species from Serbia (359.81 kcal/100 g dw). The results obtained are in agreement with
the literature, since carbohydrates and proteins are the two main components of
mushroom fruiting bodies, the first constituting about half the mushroom dry matter,19,20
proving that mushrooms could be considered valuable nutritional and healthy foods,
since they are rich in proteins and minerals and poor in calories and fat.
The free sugars composition is presented in Table 2. Free sugars composition goes
beyond its part in simply characterising the chemical constitution of the mushroom
species in question. It also provides us with some additional information which allows
us to classify the mushroom as a functional food (source of nutraceuticals –
mono/oligosaccharides). In this study, both species (from both origins) revealed the
presence of fructose, mannitol and trehalose, with no significant differences between the
total free sugars content (12.68 g/100 g dw for the Portuguese sample and 12.77 g/100 g
dw for the Serbian sample).
Mannitol, like the polyols in general, has practically no influence on blood glucose
concentrations. After polyols consumption, they remain in low concentrations in blood
because of their slow and incomplete absorption, and specifically mannitol is absorbed
and eliminated almost unchanged via the kidneys.21 Mannitol also has some medicinal
applications mainly due to its osmotic diuretic properties.22 Some bioactive effects have
been attributed to trehalose, such as suppressing the auto-oxidation of unsaturated fatty
acids.23 This capability becomes important not only from the standpoint of food
preservation but also for human health, since the oxidation products of lipids can be
detrimental and associated with the aging process.
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With these aspects in mind, mushrooms, and in this case S. granulatus, proved to be a
source of nutraceuticals, namely mono- and disaccharides as well as polyols. These
findings are in agreement with other studies from our research group which have
already identified some wild species from Portugal and Serbia as a source of such
molecules.24,25 Mannitol and trehalose are the main representatives of alcoholic sugars
and oligosaccharides usually found in mushrooms, and the mean values described in
wild mushrooms generally vary between 2.89 and 3.92 g/100 g dw, respectively.20 The
results obtained in the present work are similar to those described in the literature, since
we obtained mean values of 3.26 g/100 g dw for mannitol and 3.72 g/100 g dw for
trehalose.
Concerning the organic acid composition (Table 2), both studied species revealed no
significant differences between the total organic acids presented (4.63 g/100 g dw for
the Portuguese sample and 4.44 g/100 g dw for the Serbian sample). Nevertheless, both
profiles were somewhat different. The Portuguese sample contained oxalic acid (3.35
g/100 g dw), quinic acid (0.36 g/100 g dw) and fumaric acid (0.92 g/100 g dw). On the
other hand, the sample from Serbia was composed of oxalic acid (0.42 g/100 g dw),
malic acid (0.94 g/100 g dw), citric acid (1.77 g/100 g dw) and fumaric acid (1.31 g/100
g dw). Other studies in S. granulatus identified oxalic, aconitric, citric, malic, quinic,
succinic, shikimic and fumaric acids, with succinic and shikimic acids appearing in
lower quantities.10,26 These results support the idea that the chemical content among
species from different sources can be similar, as a characteristic of the species, but the
chemical profile may vary between them. Although morphologically similar, fungi
metabolites may be very different. Some metabolites may be produced by all the
varieties of a particular species, while others may be specific metabolites of an
organism. It should be noted that the chemistry of an organism may also vary according
to the conditions under which it develops.27 As we demonstrate, the total organic acid
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levels have no significant differences between the species, but we found different
profiles (e.g. quinic acid was only found in the Portuguese sample, while malic and
citric acids were observed in the Serbian sample). Although organic acids are
considered non-nutrients, they constitute important molecules given their biological
activities. Malic, citric, and fumaric acids, in addition to playing an important role in the
Krebs cycle which is essential for human metabolism, have many other applications.
Malic acid has been reported as having a bactericidal effect,28 being employed in food
additives as well as polymer and pharmaceutical industries.29 Citric acid (known for its
antioxidant activity) is also a crystal thickener in bones30 while fumaric acid is effective
against psoriasis and inflammation, and can be used potentially as a neuro and
chemoprotector.31,32 Due to its properties, oxalic acid constitutes part of pharmaceutical
preparations and is used for desloughing wounds and ulcers,33 while quinic acid is a
stronger antioxidant.34
Analysing the results obtained for the phenolic acids detected in the studied samples
(Table 2), we can conclude that the Portuguese sample showed a higher content of these
compounds (0.59 mg/100 g dw) compared with the Serbian samples (0.13 mg/100 g
dw). The former, was the only sample that revealed the presence of gallic acid (0.11
mg/100 g dw). On the other hand, p-hydroxybenzoic acid was present both in the
Portuguese and Serbian samples (0.48 mg/100 g dw and 0.13 mg/100 g dw,
respectively) as also the related compound cinnamic acid (0.13 mg/100 g dw and 0.03
mg/100 g dw, respectively). Phenolic acids hold antioxidant activity as chelators and
free radical scavengers with particular effects on hydroxyl and peroxyl radicals,
superoxide anions and peroxynitrites. Curiously, one of the most studied and promising
phenolic compound is gallic acid (detected in the Portuguese sample) which is a
compound belonging to the hydroxybenzoic acids group.35 Other phenolics have been
identified in S. granulatus from Portugal, namely quercetin (0.2 – 1.59 mg/100 g dw).26
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These results support the idea that since mushrooms obtain nutrients by absorption, they
are greatly influenced by the environment in which they develop, with this influence
being translated on their secondary metabolites. Given these results, we can conclude
that besides being a source of nutraceuticals, mushrooms are also a source of bioactive
compounds, namely phenolic acids. In fact, mushrooms have been described as a source
of these compounds.14,36
Analysing the results obtained for the fatty acids profile (Table 3), we can conclude that
mushrooms are a good source of “good fats”, namely mono- and polyunsaturated fatty
acids (MUFA and PUFA, respectively). Actually, these were the prevailing fatty acids
in both samples (21.30% - 64.40% of total FA). The Portuguese sample revealed a
higher content in MUFA (26.55% of total FA) while the Serbian sample showed the
highest content in PUFA (64.40% of total FA). Both samples showed a very similar
profile with the prevalence of the saturated fatty acids (SFA) palmitic acid (C16:0) and
stearic acid (C18:0), the MUFA oleic acid (C18:1n9), and the PUFA linoleic acid
(C18:2n6). These results are also in agreement with literature which reported palmitic,
oleic, linoleic, stearic and linolenic (C18:3n3) acids as the major fatty acids found in
wild mushrooms, with the latter two found in smaller percentages.19 There are studies
on the fatty acids profile of S. granulatus from Portugal.9 These authors reported that
the main fatty acids presented by the studied species were palmitic acid, palmitoleic
acid, stearic acid, oleic acid and linoleic acid; higher contents of MUFA and PUFA than
SFA were also illustrated. By comparing both studies, where the analysed species was
collected in the same region of Portugal but in different seasons/years, we can conclude
that the generic profile remains, although we can verify some fluctuations. This is
further evidence that although certain compounds may be characteristic of a particular
species, mushrooms are highly influenced by their environment (temperature, moisture,
pH). Accordingly, although palmitic acid (a nutritionally undesirable SFA) and the
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nutritional neutral saturated stearic acid are some of the mushrooms major fatty acids,
this matrix continues to be a source of nutraceuticals, as oleic and linoleic acids were
detected in higher percentages. Of interest is the observation that oleic acid has been
referred to reduce coronary heart disease risk by 20–40% mainly via LDL-cholesterol
reduction as well as having other beneficial effects on risk factors for cardiovascular
disease.37,38 Linoleic acid, an omega-6 PUFA has also been shown to reduce the risk of
coronary heart disease.39 Given the biological activities of this fatty acid found in the
studied mushroom species, we can consider them a source of molecules with health
benefits.
Concerning vitamin E, the Portuguese sample revealed the highest content of this
vitamin’s isoforms (294.94 µg/100 g dw; Table 3). This sample presented the highest
levels of α-tocopherol (17.86 µg/100 g dw) and mostly of δ-tocopherol (101.79 µg/100
g dw). β-tocopherol was the prevailing isoform in both samples, and its content was
similar between Portuguese and Serbian samples (175.29 µg/100 g dw and 179.68
µg/100 g dw, respectively). Vitamin E, as an antioxidant, exerts an important role in
lipid peroxidation. Indeed, it is the only major lipid-soluble, chain breaking antioxidant
found in plasma, red cells and tissues, allowing it to protect the integrity of lipid
structures, mainly membranes.35 Because of its function, the consumption of food where
this vitamin is present takes on added importance. Similar patterns have been detected
in different species of mushrooms both from Portugal and Serbia.13,24
Bioactive properties
Wild mushrooms have also been referred to as having bioactive properties, namely
antioxidant36 and antimicrobial40 potential. For this reason, antioxidant and
antimicrobial properties of the studied mushroom species were also evaluated.
19
The response of antioxidants to different radical or oxidant sources may differ.
Consequently, no single assay accurately reflects the mechanism of action of all radical
sources or all antioxidants in a complex system.41 Therefore, the antioxidant activity of
the studied mushrooms was assessed by resorting to five different methods (Table 4).
The Serbian sample revealed the most promising results since, in general, this sample
revealed the highest antioxidant properties. It showed the highest reducing power, with
the highest content in total phenolics assessed through the Folin-Ciocalteu assay (44.36
mg GAE/g extract) and the lowest EC50 value for the Ferricyanide/Prussian blue assay
(0.41 mg/mL). It also revealed the highest radical scavenging activity, evaluated
through the DPPH radical-scavenging activity assay (0.89 mg/mL) and the highest lipid
peroxidation inhibition assessed through the TBARS assay (0.02 mg/mL). The
exception was verified with the evaluation of the lipid peroxidation inhibition measured
through the β-carotene/linoleate assay, where both samples presented similar EC50
values with no significant differences between them (0.45 and 0.48 mg/mL). Some
studies report the antioxidant activity of S. granulatus.26 In that study, S. granulatus
revealed moderated antioxidant potential, only evaluated through the DDPH radical
scavenging activity.26
The antioxidant properties of several matrices present in the human diet, such as
mushrooms, must be assigned to the bioactive molecules obtainable from them. These
molecules include vitamins (e.g. C and E), flavonoids and other phenolic compounds, or
carotenoids.35 Although in general the Portuguese sample revealed higher levels of
vitamin E isoforms (294.94 µg/100 g dw) and phenolic acids (0.59 mg/100 g dw), the
Serbian sample showed greater antioxidant capacity. This implies that these are not the
only molecules that are contributing to the activity. Others that were not identified could
also be involved (e.g. steroids or polysaccharides).
20
Mushrooms have also been exploited as an alternative source of novel antimicrobials,
and according to the literature, mushroom extracts generally exhibit higher
antimicrobial activity against gram-positive bacteria.40 These studies are interesting, not
only from the standpoint of the discovery of new extracts/molecules with antimicrobial
potential, but also from their inclusion as food additives (preservatives), as our research
group has been demonstrating.25 Therefore, the antimicrobial potential of the studied
species can also contribute to increasing foods’ shelf life.
The results regarding the antimicrobial properties of the samples are presented in
Tables 5 and 6. Concerning the antibacterial activity (Table 5) generally, the Serbian
sample revealed better results (MIC: 0.04 – 0.15 mg/mL and MBC: 0.05 – 0.2 mg/mL).
Both samples showed bioactivity towards all the Gram positive and Gram negative
bacteria used, and in general, the values obtained were lower than those presented by
the standards streptomycin and ampicillin. Furthermore, both species also revealed
antifungal properties (Table 6) against all the strains tested. Again, the Serbian sample
registered the lowest MIC and MBC values. However, in this case, the Portuguese
sample revealed similar results for the species Aspergillus niger, Penicillium
funiculosum, P. ochrochloron and P. verrucosum var. cyclopium. Again, the values
displayed by the samples (MIC: 0.025 – 0.45 mg/mL; MFC: 0.05 – 0.8 mg/mL) were
generally lower than those of the standards (MIC: 0.1 – 1.0; MFC: 0.2 – 1.5 mg/mL).
Conclusions
This work aims to be a step forward towards classifying mushrooms, specifically Suillus
granulatus as a functional food. It provides new data concerning the chemical
characterisation of the species from the perspective of being a source of nutraceuticals
and bioactive compounds. Throughout the study, it was established that this mushroom
21
could be classified as a valuable health food, rich in carbohydrates and proteins and low
in fat. It is also an excellent source of a wide range of interesting molecules, namely
nutraceuticals such as unsaturated fatty acids, free sugars and vitamin E. S. granulatus
proved to have antioxidant and antimicrobial properties irrespective of its origin. This
way, this wild species can be consumed in either of these countries with beneficial
effects.
In conclusion, Suillus granulatus can be considered a functional food, since the
molecules found therein have, besides the nutritional effect, beneficial properties such
as antioxidant and antimicrobial activity. However, further in-depth studies such as the
study of the compounds’ mechanism of action in vitro and in vivo, need further
quantification.
Competing interests
The authors declare no competing financial interest.
Acknowledgements
The authors are grateful to Foundation for Science and Technology (FCT, Portugal) and
COMPETE/QREN/EU for the financial support of the CIMO strategic project PEst-
OE/AGR/UI0690/2011 and of the contract of L. Barros. The authors also thank to the
Serbian Ministry of Education, Science and Technological Development for financial
support (grant number 173032). The authors thank Dr. Maria João Sousa for the harvest
of the Portuguese samples.
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25
Table 1. Nutritional value (mean ± SD).
Different letters in each row indicate significant differences between the samples (p<0.05). dw- dry weight.
Suillus granulatus (L.) Roussel
Portugal Serbia
Ash (g/100 g dw) 7.99 ± 0.91b 10.38 ± 0.08a
Proteins (g/100 g dw) 14.78 ± 0.41a 7.93 ± 0.00b
Fat (g/100 g dw) 3.74 ± 0.20a 0.27 ± 0.09b
Carbohydrates (g/100 g dw) 73.49 ± 0.46b 81.42 ± 0.01a
Energy (kcal/100 g dw) 386.74 ± 3.26a 359.81 ± 0.58b
26
Table 2. Hydrophilic compounds (mean ± SD).
Different letters in each row indicate significant differences between the samples (p<0.05). dw- dry weight; nd- not detected.
Suillus granulatus (L.) Roussel
Portugal Serbia
Fructose (g/100g dw) 4.49 ± 0.02b 7.02 ± 0.16a
Mannitol (g/100g dw) 3.33 ± 0.10a 3.18 ± 0.14a
Trehalose (g/100g dw) 4.86 ± 0.06a 2.57 ± 0.13b
Total frees sugars (g/100g dw) 12.68 ± 0.01a 12.77 ± 0.42a
Oxalic acid (g/100 g dw) 3.35 ± 0.16a 0.42 ± 0.02b
Quinic acid (g/100 g dw) 0.36 ± 0.02 nd
Malic acid (g/100 g dw) nd 0.94 ± 0.16
Citric acid (g/100 g dw) nd 1.77 ± 0.25
Fumaric acid (g/100 g dw) 0.92 ± 0.00b 1.31 ± 0.03a
Total organic acids (g/100 g dw) 4.63 ± 0.14a 4.44 ± 0.09a
Gallic acid (mg/100 g dw) 0.11 ± 0.01 nd
p-Hydroxybenzoic acid (mg/100 g dw) 0.48 ± 0.00a 0.13 ± 0.01b
Total phenolic acids (mg/100 g dw) 0.59 ± 0.01a 0.13 ± 0.01b
Cinnamic acid (mg/100 g dw) 0.13 ± 0.00a 0.03 ± 0.00b
27
Table 3. Lipophilic compounds (mean ± SD).
Suillus granulatus (L.) Roussel Fatty acids Portugal Serbia C6:0 0.02 ± 0.00 0.05 ± 0.00 C8:0 0.02 ± 0.01 0.11 ± 0.00 C10:0 0.05 ± 0.01 0.04 ± 0.00 C12:0 0.03 ± 0.00 0.03 ± 0.00 C13:0 0.01 ± 0.00 nd C14:0 0.20 ± 0.01 0.17 ± 0.00 C14:1 0.02 ± 0.00 tr C15:0 0.80 ± 0.01 0.58 ± 0.01 C16:0 9.64 ± 0.04 9.62 ± 0.13 C16:1 0.46 ± 0.03 0.49 ± 0.01 C17:0 0.24 ± 0.00 0.19 ± 0.00 C18:0 3.19 ± 0.01 2.65 ± 0.15 C18:1n9 24.64 ± 0.14 20.08 ± 0.16 C18:2n6 57.14 ± 0.19 63.97 ± 0.46 C18:3n3 0.39 ± 0.01 0.20 ± 0.00 C20:0 0.28 ± 0.02 0.16 ± 0.00 C20:1 0.15 ± 0.04 0.13 ± 0.00 C20:2 0.18 ± 0.03 0.15 ± 0.00 C20:3n3+C21:0 0.16 ± 0.01 0.08 ± 0.00 C20:5n3 0.14 ± 0.01 0.01 ± 0.00 C22:0 0.40 ± 0.04 0.29 ± 0.00 C22:1n9 0.44 ± 0.03 0.21 ± 0.00 C23:0 0.09 ± 0.01 0.11 ± 0.00 C24:0 0.47 ± 0.09 0.32 ± 0.00 C24:1 0.84 ± 0.09 0.38 ± 0.00 Total SFA (% of total FA) 15.44 ± 0.10a 14.30 ± 0.29b Total MUFA (% of total FA) 26.55 ± 0.05a 21.30 ± 0.17b Total PUFA (% of total FA) 58.01 ± 0.15b 64.40 ± 0.46a α-Tocopherol (µg/100g dw) 17.86 ± 1.07a 6.81 ± 0.40b β-Tocopherol (µg/100g dw) 175.29 ± 4.02a 179.68 ± 0.90a γ-Tocopherol (µg/100g dw) nd 13.61 ± 1.40 δ-Tocopherol (µg/100g dw) 101.79 ± 7.04a 19.82 ± 0.40b Total tocopherols (µg/100g dw) 294.94 ± 9.99a 219.92 ± 1.31b
Different letters in each row indicate significant differences between the samples (p<0.05). Caproic acid (C6:0); Caprylic acid (C8:0); Capric acid (C10:0); Lauric acid (C12:0); Tridecylic acid (C13:0); Myristic acid (C14:0); Myristoleic acid (C14:1); Pentadecanoic acid (C15:0); Palmitic acid (C16:0); Palmitoleic acid (C16:1); Heptadecanoic acid (C17:0); Stearic acid (C18:0); Oleic acid (C18:1n9); Linoleic acid (C18:2n6); α-Linolenic acid (C18:3n3); Arachidic acid (C20:0); Eicosenoic acid (C20:1); cis-11,14-Eicosadienoic acid (C20:2); cis-11,14,17-Eicosatrienoic acid and Heneicosanoic acid (C20:3n3+C21:0); cis-5,8,11,14,17-Eicosapentaenoic acid (C20:5n3); Behenic acid (C22:0); Behenic acid (C22:1n9); Tricosanoic acid (C23:0); Lignoceric acid (C24:0); Nervonic acid (C24:1). SFA- saturated fatty acids;
28
MUFA- monounsaturated fatty acids; PUFA- polyunsaturated fatty acids. dw- dry weight; nd- not detected; tr- traces.
29
Table 4. Antioxidant properties of the methanolic extracts (mean ± SD).
Different letters in each row indicate significant differences between the extracts (p<0.05). Concerning the Folin-Ciocalteu assay, higher values mean higher reducing power; for the other assays, the results are presented in EC50 values, what means that higher values correspond to lower reducing power or antioxidant potential. EC50: Extract concentration corresponding to 50% of antioxidant activity or 0.5 of absorbance for the Ferricyanide/Prussian blue assay.
Suillus granulatus (L.) Roussel methanolic extracts
Activity Assay Portugal Serbia
Reducing Power Folin-Ciocalteu assay (mg GAE/g extract) 40.78 ± 0.88b 44.36 ± 0.31a
Ferricyanide/Prussian blue assay (EC50; mg/mL) 0.57 ± 0.01a 0.41 ± 0.01b
Radical scavenging activity DPPH radical-scavenging activity assay (EC50; mg/mL) 0.98 ± 0.02a 0.89 ± 0.02b
Lipid peroxidation
inhibition
β-carotene/linoleate assay (EC50; mg/mL) 0.45 ± 0.08a 0.48 ± 0.06a
TBARS assay (EC50; mg/mL) 0.03 ± 0.00a 0.02 ± 0.01b
30
Table 5. Antibacterial properties of the methanolic extracts (mg/mL; mean ± SD).
Different letters in each row indicate significant differences between the extracts (p<0.05). MIC- minimum inhibitory concentration; MBC- minimum bactericidal concentration.
Suillus granulatus (L.) Roussel methanolic extracts Portugal Serbia Streptomycin Ampicillin
Staphylococcus aureus MIC 0.15 ± 0.007b 0.05 ± 0.00c 0.04 ± 0.002d 0.25 ± 0.00a MBC 0.2 ± 0.02b 0.1 ± 0.007c 0.09 ± 0.003c 0.37 ± 0.007ª
Bacillus cereus MIC 0.1 ± 0.02b 0.04 ± 0.001c 0.09 ± 0.000b 0.25 ± 0.02ª MBC 0.2 ± 0.03b 0.05 ± 0.00c 0.17 ± 0.007b 0.37 ± 0.02ª
Micrococcus flavus MIC 0.2 ± 0.02ª 0.1 ± 0.02b 0.17 ± 0.007b 0.25 ± 0.02ª MBC 0.4 ± 0.07ª 0.2 ± 0.02c 0.34 ± 0.003b 0.37 ± 0.01ba
Listeria monocytogenes MIC 0.2 ± 0.00b 0.05 ± 0.03c 0.17 ± 0.01b 0.37 ± 0.009ª MBC 0.4 ± 0.003b 0.2 ± 0.000d 0.34 ± 0.01c 0.49 ± 0.003ª
Pseudomonas aeruginosa MIC 0.15 ± 0.02b 0.05 ± 0.007c 0.17 ± 0.007b 0.74 ± 0.006ª MBC 0.2 ± 0.00c 0.1 ± 0.02d 0.34 ± 0.003b 1.24 ± 0.02ª
Salmonella typhimurium MIC 0.15 ± 0.007c 0.05 ± 0.00d 0.17 ± 0.007b 0.37 ± 0.007ª MBC 0.2 ± 0.01c 0.1 ± 0.000d 0.34 ± 0.003b 0.49 ± 0.01ª
Escherichia coli MIC 0.15 ± 0.00cb 0.15 ± 0.02c 0.17 ± 0.01b 0.25 ± 0.00a MBC 0.2 ± 0.01c 0.2 ± 0.00d 0.34 ± 0.003b 0.49 ± 0.01ª
Enterobacter cloacae MIC 0.15 ± 0.03c 0.1 ± 0.00d 0.26 ± 0.006b 0.37 ± 0.007ª
MBC 0.2 ± 0.01c 0.2 ± 0.007c 0.52 ± 0.007b 0.74 ± 0.003a
31
Table 6. Antifungal properties of the methanolic extracts (mg/mL; mean ± SD).
Different letters in each row indicate significant differences between the extracts (p<0.05). MIC- minimum inhibitory concentration; MFC- minimum fungicidal concentration.
Suillus granulatus (L.) Roussel methanolic extracts
Portugal Serbia Ketoconazole Bifonazole
Aspergillus fumigatus MIC 0.45 ± 0.02a 0.05 ± 0.00d 0.2 ± 0.01b 0.15 ± 0.02c
MFC 0.8 ± 0.02a 0.2 ± 0.02c 0.5 ± 0.00b 0.2 ± 0.01c
Aspergillus versicolor MIC 0.1 ± 0.01b 0.025 ± 0.0007c 0.2 ± 0.02a 0.1 ± 0.02b MFC 0.2 ± 0.02b 0.1 ± 0.01c 0.5 ± 0.02a 0.2 ± 0.01b
Aspergillus ochraceus MIC 0.1 ± 0.00b 0.025 ± 0.002c 0.15 ± 0.02a 0.15 ± 0.007a MFC 0.2 ± 0.00a 0.05 ± 0.007b 0.2 ± 0.01a 0.2 ± 0.01a
Aspergillus niger MIC 0.05 ± 0.003c 0.05 ± 0.01c 0.2 ± 0.00a 0.15 ± 0.001b MFC 0.1 ± 0.02c 0.1 ± 0.00c 0.5 ± 0.02a 0.2 ± 0.007b
Trichoderma víride MIC 0.075 ± 0.008b 0.01 ± 0.00c 1.0 ± 0.07a 0.15 ± 0.02b MFC 0.1 ± 0.01cb 0.05 ± 0.00c 1.5 ± 0.10a 0.2 ± 0.02b
Penicillium funiculosum MIC 0.05 ± 0.007b 0.05 ± 0.01b 0.2 ± 0.02a 0.2 ± 0.00a MFC 0.1 ± 0.02c 0.1 ± 0.00c 0.5 ± 0.02a 0.25 ± 0.02b
Penicillium ochrochloron MIC 0.075 ± 0.008c 0.05 ± 0.00c 1.0 ± 0.07a 0.2 ± 0.01b MFC 0.1 ± 0.01c 0.2 ± 0.007cb 1.5 ± 0.07a 0.25 ± 0.01b
Penicillium verrucosum var. cyclopium MIC 0.1 ± 0.01b 0.1 ± 0.02b 1.5 ± 0.07a 0.2 ± 0.00b MFC 0.4 ± 0.03b 0.2 ± 0.02b 2.0 ± 0.10a 0.3 ± 0.02b